Recombinant virus products and methods for inhibition of expression of myotilin

ABSTRACT

The present invention relates to RNA interference-based methods for inhibiting the expression of the myotilin gene. Recombinant adeno-associated viruses of the invention deliver DNAs encoding microRNAs that knock down the expression of myotilin. The methods have application in the treatment of muscular dystrophies such as Limb Girdle Muscular Dystrophy Type 1A.

This application is a continuation-in-part of International ApplicationNo. PCT/US/2012/034408 filed Apr. 20, 2012 which in turn is acontinuation-in-part of U.S. Provisional Patent Application No.61/478,012 filed Apr. 21, 2011, which documents are incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to RNA interference-based methods forinhibiting the expression of the myotilin gene. Recombinantadeno-associated viruses of the invention deliver DNAs encodingmicroRNAs that knock down the expression of myotilin. The methods haveapplication in the treatment of muscular dystrophies such as Limb GirdleMuscular Dystrophy Type 1A.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

This application contains, as a separate part of disclosure, a SequenceListing in computer-readable form (filename: 46208PCT_SeqListing.txt;1,721,237 bytes—ASCII text file) which is incorporated by referenceherein in its entirety.

BACKGROUND

Muscular dystrophies (MDs) are a group of genetic diseases. The group ischaracterized by progressive weakness and degeneration of the skeletalmuscles that control movement. Some forms of MD develop in infancy orchildhood, while others may not appear until middle age or later. Thedisorders differ in terms of the distribution and extent of muscleweakness (some forms of MD also affect cardiac muscle), the age ofonset, the rate of progression, and the pattern of inheritance.

One group of MDs is the limb girdle group (LGMD) of MDs. LGMDs are rareconditions and they present differently in different people with respectto age of onset, areas of muscle weakness, heart and respiratoryinvolvement, rate of progression and severity. LGMDs can begin inchildhood, adolescence, young adulthood or even later. Both genders areaffected equally. LGMDs cause weakness in the shoulder and pelvicgirdle, with nearby muscles in the upper legs and arms sometimes alsoweakening with time. Weakness of the legs often appears before that ofthe arms. Facial muscles are usually unaffected. As the conditionprogresses, people can have problems with walking and may need to use awheelchair over time. The involvement of shoulder and arm muscles canlead to difficulty in raising arms over head and in lifting objects. Insome types of LGMD, the heart and breathing muscles may be involved.

There are at least nineteen forms of LGMD, and the forms are classifiedby their associated genetic defects.

Type Pattern of Inheritance Gene or Chromosome LGMD1A Autosomal dominantMyotilin gene LGMD1B Autosomal dominant Lamin A/C gene LGMD1C Autosomaldominant Caveolin gene LGMD1D Autosomal dominant Chromosome 7 LGMD1EAutosomal dominant Desmin gene LGMD1F Autosomal dominant Chromosome 7LGMD1G Autosomal dominant Chromosome 4 LGMD2A Autosomal recessiveCalpain-3 gene LGMD2B Autosomal recessive Dysferlin gene LGMD2CAutosomal recessive Gamma-sarcoglycan gene LGMD2D Autosomal recessiveAlpha-sarcoglycan gene LGMD2E Autosomal recessive Beta-sarcoglycan geneLGMD2F Autosomal recessive Delta-sarcoglycan gene LGMD2G Autosomalrecessive Telethonin gene LGMD2H Autosomal recessive TRIM32 LGMD2IAutosomal recessive FKRP gene LGMD2J Autosomal recessive Titin geneLGMD2K Autosomal recessive POMT1 gene LGMD2L Autosomal recessive Fukutingene

Specialized tests for LGMD are now available through a national schemefor diagnosis, the National Commissioning Group (NCG).

LGMD1A is caused by gain-of-function missense mutations in the myotilin(MYOT) gene [Hauser et al., Am. J. Hum. Genet, 71: 1428-1432 (2002);Hauser et al., Hum. Mol. Genet., 9: 2141-2147 (2000); Shalaby et al., J.Neuropathol. Exp. Neurol., 68: 701-707 (2009)]. LGMD1A patients developproximal leg and arm weakness in early adulthood (25 years is mean onsetage), which progresses to the distal limb musculature. At thehistological level, patients show myofiber degeneration and sizevariability, fiber splitting, centrally located myonuclei, autophagicvesicles, and replacement of myofibers with fat and fibrotic tissue,which are all common features of muscular dystrophy. Patients withLGMD1A also develop intramuscular myofibrillar protein aggregates,rimmed vacuoles, and severe Z-disc disorganization (called Z-discstreaming), which completely disrupt the sarcomeric structure. Atransgenic mouse model, the T571 mouse model, using a mutant human MYOTallele has been developed [Garvey et al., Hum. Mol. Genet. 15: 2348-2362(2006)]. Importantly, T57I mice recapitulate the progressivehistological and functional abnormalities associated with LGMD1A,including reduced muscle size, muscle weakness, intramuscularmyofibrillar aggregates, Z-disc streaming, and centrally locatedmyonuclei.

The myotilin gene encodes a 57 kDa protein expressed primarily inskeletal and cardiac muscle. Myotilin appears to function as astructural component of the Z-disc, and may therefore contribute tosarcomere assembly, actin filament stabilization, and force transmissionin striated muscle. Nevertheless, myotilin is not required for normalmuscle development or function, since myotilin null mice are overtly andhistologically normal. Specifically, mouse muscles lacking myotilin areindistinguishable from wild type in muscle mass, myofiber size,contractile strength (specific force), and sarcolemmal integrity.Moreover, MYOT null mice develop normally, live a normal life span, andshow no histological evidence of muscular dystrophy or Z-discmalformations. Mouse and human myotilin transcripts are expressed in thesame tissues, have the same genomic structures, and protein sequencesare highly conserved (90% identity; 94% similarity), which indicates aconserved functional.

RNA interference (RNAi) is a mechanism of gene regulation in eukaryoticcells that has been considered for the treatment of various diseases.RNAi refers to post-transcriptional control of gene expression mediatedby microRNAs (miRNAs). The miRNAs are small (21-25 nucleotides),noncoding RNAs that share sequence homology and base-pair with cognatemessenger RNAs (mRNAs). The interaction between the miRNAs and mRNAsdirects cellular gene silencing machinery to prevent the translation ofthe mRNAs. The RNAi pathway is summarized in Duan (Ed.), Section 7.3 ofChapter 7 in Muscle Gene Therapy, Springer Science+Business Media, LLC(2010). Section 7.4 mentions MYOT RNAi therapy of LGMD 1A in mice todemonstrate proof-of-principle for RNAi therapy of dominant muscledisorders.

As an understanding of natural RNAi pathways has developed, researchershave designed artificial miRNAs for use in regulating expression oftarget genes for treating disease. As described in Section 7.4 of Duan,supra, artificial miRNAs can be transcribed from DNA expressioncassettes. The miRNA sequence specific for a target gene is transcribedalong with sequences required to direct processing of the miRNA in acell. Viral vectors such as adeno-associated virus have been used todeliver miRNAs to muscle [Fechner et al., J. Mol. Med., 86: 987-997(2008).

Adeno-associated virus (AAV) is a replication-deficient parvovirus, thesingle-stranded DNA genome of which is about 4.7 kb in length including145 nucleotide inverted terminal repeat (ITRs). There are multipleserotypes of AAV. The nucleotide sequences of the genomes of the AAVserotypes are known. For example, the complete genome of AAV-1 isprovided in GenBank Accession No. NC_(—)002077; the complete genome ofAAV-2 is provided in GenBank Accession No. NC_(—)001401 and Srivastavaet al., J. Virol., 45: 555-564 {1983); the complete genome of AAV-3 isprovided in GenBank Accession No. NC_(—)1829; the complete genome ofAAV-4 is provided in GenBank Accession No. NC_(—)001829; the AAV-5genome is provided in GenBank Accession No. AF085716; the completegenome of AAV-6 is provided in GenBank Accession No. NC_(—)00 1862; atleast portions of AAV-7 and AAV-8 genomes are provided in GenBankAccession Nos. AX753246 and AX753249, respectively; the AAV-9 genome isprovided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11genome is provided in Virology, 330(2): 375-383 (2004). Cis-actingsequences directing viral DNA replication (rep), encapsidation/packagingand host cell chromosome integration are contained within the AAV ITRs.Three AAV promoters (named p5, p19, and p40 for their relative maplocations) drive the expression of the two AAV internal open readingframes encoding rep and cap genes. The two rep promoters (p5 and p19),coupled with the differential splicing of the single AAV intron (atnucleotides 2107 and 2227), result in the production of four repproteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Repproteins possess multiple enzymatic properties that are ultimatelyresponsible for replicating the viral genome. The cap gene is expressedfrom the p40 promoter and it encodes the three capsid proteins VP1, VP2,and VP3. Alternative splicing and non-consensus translational startsites are responsible for the production of the three related capsidproteins. A single consensus polyadenylation site is located at mapposition 95 of the AAV genome. The life cycle and genetics of AAV arereviewed in Muzyczka, Current Topics in Microbiology and Immunology,158: 97-129 (1992).

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA to cells, for example, in gene therapy. AAVinfection of cells in culture is noncytopathic, and natural infection ofhumans and other animals is silent and asymptomatic. Moreover, AAVinfects many mammalian cells allowing the possibility of targeting manydifferent tissues in vivo. Moreover, AAV transduces slowly dividing andnon-dividing cells, and can persist essentially for the lifetime ofthose cells as a transcriptionally active nuclear episome(extrachromosomal element). The AAV proviral genome is infectious ascloned DNA in plasmids which makes construction of recombinant genomesfeasible. Furthermore, because the signals directing AAV replication,genome encapsidation and integration are contained within the ITRs ofthe AAV genome, some or all of the internal approximately 4.3 kb of thegenome (encoding replication and structural capsid proteins, rep-cap)may be replaced with foreign DNA. The rep and cap proteins may beprovided in trans. Another significant feature of AAV is that it is anextremely stable and hearty virus. It easily withstands the conditionsused to inactivate adenovirus (56° to 65° C. for several hours), makingcold preservation of AAV less critical. AAV may even be lyophilized.Finally, AAV-infected cells are not resistant to superinfection.

There remains a need in the art for a treatment for LGMD1A.

SUMMARY

The present invention provides methods and products for preventing orinhibiting the expression of the MYOT gene. The methods of the inventionutilize RNAi to prevent or inhibit the expression of the MYOT gene. Themethods involve delivering inhibitory RNAs specific for the MYOT gene tomuscle cells. The MYOT inhibitory RNAs contemplated include, but are notlimited to, antisense RNAs, small inhibitory RNAs (siRNAs), shorthairpin RNAs (shRNAs) or artificial microRNAs (MYOT miRNAs) that inhibitexpression of MYOT. Use of the methods and products is indicated, forexample, in preventing or treating LGMD1A. Some embodiments of theinvention exploit the unique properties of AAV to deliver DNA encodingMYOT inhibitory RNAs to muscle cells. Other embodiments of the inventionutilize other vectors (for example, other viral vectors such asadenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus,pox viruses, herpes virus, polio virus, sindbis virus and vacciniaviruses) to deliver polynucleotides encoding MYOT inhibitory RNAs.

In one aspect, the invention provides MYOT miRNAs. In another aspect,the invention provides rAAV encoding the MYOT miRNAs wherein the rAAVlack rep and cap genes. In some embodiments, the MYOT miRNA comprises anmiRNA antisense guide strand selected from those set out in SEQ ID NO: 7through SEQ ID NO: 11266. These sequences comprise antisense “guide”strand sequences of the invention of varying sizes. The antisense guidestrand is the strand of the mature miRNA duplex that becomes the RNAcomponent of the RNA induced silencing complex ultimately responsiblefor sequence-specific gene silencing. See Section 7.3 of Duan, supra.For example, the first antisense guide strand in SEQ ID NO: 7corresponds to (is the reverse complement of) the 3′ end of the myotilinsequence set out in FIG. 1. The second antisense guide strand (SEQ IDNO: 8) is offset one nucleotide from the first and so on. In someembodiments, the GC content of the antisense guide strand is 60% orless, and/or the 5′ end of the antisense guide strand is more AU richwhile the 3′ end is more GC rich. Exemplified MYOT miRNA are encoded bythe DNAs set out in SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, rAAVare self-complementary (sc) AAV. In some embodiments, the MYOT miRNAencoding sequences are under the control of a muscle-specific tMCK orCK6 promoter.

In another aspect, the invention provides a composition comprising therAAV encoding the MYOT miRNA.

In yet another aspect, the invention provides a method of preventing orinhibiting expression of the MYOT gene in a cell comprising contactingthe cell with a rAAV encoding an MYOT miRNA, wherein the miRNA isencoded by the DNA set out in SEQ ID NO: 1, 2, 3 or 4 and wherein therAAV lacks rep and cap genes. Expression of MYOT is inhibited by atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 or 99 percent.

In still another aspect, the invention provides a method of deliveringDNA encoding the MYOT miRNA set out in SEQ ID NO: 1, 2, 3 or 4 to ananimal in need thereof, comprising respectively administering to theanimal a rAAV encoding the MYOTmi RNA, wherein the rAAV lacks rep andcap genes.

In yet another aspect, the invention provides a method of preventing ortreating a musclular dystrophy (including, but not limited to, LGMD1A)comprising administering a rAAV encoding an MYOT miRNA, wherein themiRNA is encoded by the DNA set out in SEQ ID NO: 1, 2, 3 or 4 andwherein the rAAV lacks rep and cap genes. “Treating” may includeameliorating one or more symptoms of the muscular dystrophy (such asLGMD1A). Molecular, biochemical, histological, and functional endpointsdemonstrate the therapeutic efficacy of MYOT miRNAs. Endpointscontemplated by the invention include one or more of: the reduction orelimination of mutant MYOT protein in affected muscles, MYOT geneknockdown, reduction or elimination of (for example, LGMD1A-associated)pathogenic protein aggregates in muscle, increase in myofiber diameters,and improvement in muscle strength.

DETAILED DESCRIPTION

Recombinant AAV genomes of the invention comprise one or more AAV ITRsflanking a polynucleotide encoding, for example, one or more MYOTmiRNAs. The polynucleotide is operatively linked to transcriptionalcontrol DNA, specifically promoter DNA that is functional in target.Commercial providers such as Ambion Inc. (Austin, Tex.), Darmacon Inc.(Lafayette, Colo.), InvivoGen (San Diego, Calif.), and MolecularResearch Laboratories, LLC (Herndon, Va.) generate custom inhibitory RNAmolecules. In addition, commercially kits are available to producecustom siRNA molecules, such as SILENCER™ siRNA Construction Kit (AmbionInc., Austin, Tex.) or psiRNA System (InvivoGen, San Diego, Calif.).Embodiments include a rAAV genome comprising: the DNA set out in SEQ IDNO: 1 encoding the MYOT miRNA (named “miMyoT-1291”), the DNA set out inSEQ ID NO: 2 encoding the MYOT miRNA (named “miMyoT-1321”), the DNA setout in SEQ ID NO: 3 encoding the MYOT miRNA (named “miMyoT-1366”) or theDNA set out in SEQ ID NO: 4 encoding the MYOT miRNA (named“miMyoT-1490”).

The rAAV genomes of the invention lack AAV rep and cap DNA. AAV DNA inthe rAAV genomes may be from any AAV serotype for which a recombinantvirus can be derived including, but not limited to, AAV serotypes AAV-1,AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 andAAV-11. As noted in the Background section above, the nucleotidesequences of the genomes of various AAV serotypes are known in the art.

DNA plasmids of the invention comprise rAAV genomes of the invention.The DNA plasmids are transferred to cells permissible for infection witha helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus orherpesvirus) for assembly of the rAAV genome into infectious viralparticles. Techniques to produce rAAV particles, in which an AAV genometo be packaged, rep and cap genes, and helper virus functions areprovided to a cell are standard in the art. Production of rAAV requiresthat the following components are present within a single cell (denotedherein as a packaging cell): a rAAV genome, AAV rep and cap genesseparate from (i.e., not in) the rAAV genome, and helper virusfunctions. The AAV rep and cap genes may be from any AAV serotype forwhich recombinant virus can be derived and may be from a different AAVserotype than the rAAV genome ITRs, including, but not limited to, AAVserotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9,AAV-10 and AAV-11. Production of pseudotyped rAAV is disclosed in, forexample, WO 01/83692 which is incorporated by reference herein in itsentirety.

A method of generating a packaging cell is to create a cell line thatstably expresses all the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senaphthy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line is then infected with a helpervirus such as adenovirus. The advantages of this method are that thecells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus rather than plasmids to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/U598/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. No. 5,786,211; U.S. Pat.No. 5,871,982; U.S. Pat. No. 6,258,595; and McCarty, Mol. Ther., 16(10):1648-1656 (2008). The foregoing documents are hereby incorporated byreference in their entirety herein, with particular emphasis on thosesections of the documents relating to rAAV production. The productionand use of sc rAAV are specifically contemplated.

The invention thus provides packaging cells that produce infectiousrAAV. In one embodiment packaging cells may be stably transformed cancercells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293line). In another embodiment, packaging cells are cells that are nottransformed cancer cells, such as low passage 293 cells (human fetalkidney cells transformed with E1 of adenovirus), MRC-5 cells (humanfetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells(monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

Recombinant AAV (i.e., infectious encapsidated rAAV particles) of theinvention comprise a rAAV genome. Embodiments include, but are notlimited to, the rAAV including a genome encoding the MYOT miRNA set outin SEQ ID NO: 1(named “AAV-U6-miMyoT-1291”), the rAAV including a genomeencoding the MYOT miRNA set out in SEQ ID NO: 2 (named“AAV-U6-miMyoT-1321”), the rAAV including a genome encoding the MYOTmiRNA set out in SEQ ID NO: 3 (named “AAV-U6-miMyoT-1366”) and the rAAVincluding a genome encoding the MYOT miRNA set out in SEQ ID NO: 4(named “AAV-U6-miMyoT-1490”). The genomes of the rAAV lack AAV rep andcap DNA, that is, there is no AAV rep or cap DNA between the ITRs of thegenomes.

The rAAV may be purified by methods standard in the art such as bycolumn chromatography or cesium chloride gradients. Methods forpurifying rAAV vectors from helper virus are known in the art andinclude methods disclosed in, for example, Clark et al., Hum. GeneTher., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98/09657.

In another embodiment, the invention contemplates compositionscomprising rAAV of the present invention. Compositions of the inventioncomprise rAAV in a pharmaceutically acceptable carrier. The compositionsmay also comprise other ingredients such as diluents and adjuvants.Acceptable carriers, diluents and adjuvants are nontoxic to recipientsand are preferably inert at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, or other organic acids;antioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics or polyethylene glycol (PEG).

Titers of rAAV to be administered in methods of the invention will varydepending, for example, on the particular rAAV, the mode ofadministration, the treatment goal, the individual, and the cell type(s)being targeted, and may be determined by methods standard in the art.Titers of rAAV may range from about 1×10⁶, about 1×10⁷, about 1×10⁸,about 1×10⁹, about 1×10¹⁰, about 1×10¹¹, about 1×10¹², about 1×10¹³ toabout 1×10¹⁴ or more DNase resistant particles (DRP) per ml. Dosages mayalso be expressed in units of viral genomes (vg).

Methods of transducing a target cell with rAAV, in vivo or in vitro, arecontemplated by the invention. The in vivo methods comprise the step ofadministering an effective dose, or effective multiple doses, of acomposition comprising a rAAV of the invention to an animal (including ahuman being) in need thereof. If the dose is administered prior todevelopment of a disorder/disease, the administration is prophylactic.If the dose is administered after the development of a disorder/disease,the administration is therapeutic. In embodiments of the invention, aneffective dose is a dose that alleviates (eliminates or reduces) atleast one symptom associated with the disorder/disease state beingtreated, that slows or prevents progression to a disorder/disease state,that slows or prevents progression of a disorder/disease state, thatdiminishes the extent of disease, that results in remission (partial ortotal) of disease, and/or that prolongs survival. An example of adisease contemplated for prevention or treatment with methods of theinvention is LGMD1A.

Combination therapies are also contemplated by the invention.Combination as used herein includes both simultaneous treatment orsequential treatments. Combinations of methods of the invention withstandard medical treatments (e.g., corticosteroids) are specificallycontemplated, as are combinations with novel therapies.

Administration of an effective dose of the compositions may be by routesstandard in the art including, but not limited to, intramuscular,parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial,intraosseous, intraocular, rectal, or vaginal. Route(s) ofadministration and serotype(s) of AAV components of the rAAV (inparticular, the AAV ITRs and capsid protein) of the invention may bechosen and/or matched by those skilled in the art taking into accountthe infection and/or disease state being treated and the targetcells/tissue(s) that are to express the MYOT miRNAs.

In particular, actual administration of rAAV of the present inventionmay be accomplished by using any physical method that will transport therAAV recombinant vector into the target tissue of an animal.Administration according to the invention includes, but is not limitedto, injection into muscle, the bloodstream and/or directly into theliver. Simply resuspending a rAAV in phosphate buffered saline has beendemonstrated to be sufficient to provide a vehicle useful for muscletissue expression, and there are no known restrictions on the carriersor other components that can be co-administered with the rAAV (althoughcompositions that degrade DNA should be avoided in the normal mannerwith rAAV). Capsid proteins of a rAAV may be modified so that the rAAVis targeted to a particular target tissue of interest such as muscle.See, for example, WO 02/053703, the disclosure of which is incorporatedby reference herein. Pharmaceutical compositions can be prepared asinjectable formulations or as topical formulations to be delivered tothe muscles by transdermal transport. Numerous formulations for bothintramuscular injection and transdermal transport have been previouslydeveloped and can be used in the practice of the invention. The rAAV canbe used with any pharmaceutically acceptable carrier for ease ofadministration and handling.

For purposes of intramuscular injection, solutions in an adjuvant suchas sesame or peanut oil or in aqueous propylene glycol can be employed,as well as sterile aqueous solutions. Such aqueous solutions can bebuffered, if desired, and the liquid diluent first rendered isotonicwith saline or glucose. Solutions of rAAV as a free acid (DNA containsacidic phosphate groups) or a pharmacologically acceptable salt can beprepared in water suitably mixed with a surfactant such ashydroxypropylcellulose. A dispersion of rAAV can also be prepared inglycerol, liquid polyethylene glycols and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms. In thisconnection, the sterile aqueous media employed are all readilyobtainable by standard techniques well-known to those skilled in theart.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating actions of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of a dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating rAAV in therequired amount in the appropriate solvent with various otheringredients enumerated above, as required, followed by filtersterilization. Generally, dispersions are prepared by incorporating thesterilized active ingredient into a sterile vehicle which contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying technique that yield a powder of theactive ingredient plus any additional desired ingredient from thepreviously sterile-filtered solution thereof.

Transduction with rAAV may also be carried out in vitro. In oneembodiment, desired target muscle cells are removed from the subject,transduced with rAAV and reintroduced into the subject. Alternatively,syngeneic or xenogeneic muscle cells can be used where those cells willnot generate an inappropriate immune response in the subject.

Suitable methods for the transduction and reintroduction of transducedcells into a subject are known in the art. In one embodiment, cells canbe transduced in vitro by combining rAAV with muscle cells, e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest using conventional techniques such as Southern blots and/orPCR, or by using selectable markers. Transduced cells can then beformulated into pharmaceutical compositions, and the compositionintroduced into the subject by various techniques, such as byintramuscular, intravenous, subcutaneous and intraperitoneal injection,or by injection into smooth and cardiac muscle, using e.g., a catheter.

Transduction of cells with rAAV of the invention results in sustainedexpression of MYOT miRNAs. The present invention thus provides methodsof administering/delivering rAAV which express MYOT miRNAs to an animal,preferably a human being. These methods include transducing tissues(including, but not limited to, tissues such as muscle, organs such asliver and brain, and glands such as salivary glands) with one or morerAAV of the present invention. Transduction may be carried out with genecassettes comprising tissue specific control elements. For example, oneembodiment of the invention provides methods of transducing muscle cellsand muscle tissues directed by muscle specific control elements,including, but not limited to, those derived from the actin and myosingene families, such as from the myoD gene family [See Weintraub et al.,Science, 251: 761-766 (1990], the myocyte-specific enhancer bindingfactor MEF-2 [Cserjesi and Olson, Mol Cell Biol 11: 4854-4862 (1990],control elements derived from the human skeletal actin gene [Muscat etal., Mol Cell Biol, 7: 4089-4099 (1987)], the cardiac actin gene, musclecreatine kinase sequence elements [See Johnson et al., Mol Cell Biol,9:3393-3399 (1989)] and the murine creatine kinase enhancer (mCK)element, control elements derived from the skeletal fast-twitch troponinC gene, the slow-twitch cardiac troponin C gene and the slow-twitchtroponin I gene: hypozia-inducible nuclear factors [Semenza et al., ProcNatl Acad Sci USA, 88: 5680-5684 (1990], steroid-inducible elements andpromoters including the glucocorticoid response element (GRE) [See Maderand White, Proc. Natl. Acad. Sci. USA, 90: 5603-5607 (1993)], the tMCKpromoter [see Wang et al., Gene Therapy, 15: 1489-1499 (2008)], the CK6promoter [see Wang et al., supra] and other control elements.

Muscle tissue is an attractive target for in vivo DNA delivery, becauseit is not a vital organ and is easy to access. The inventioncontemplates sustained expression of miRNAs from transduced myofibers.

By “muscle cell” or “muscle tissue” is meant a cell or group of cellsderived from muscle of any kind (for example, skeletal muscle and smoothmuscle, e.g. from the digestive tract, urinary bladder, blood vessels orcardiac tissue). Such muscle cells may be differentiated orundifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytesand cardiomyoblasts.

The term “transduction” is used to refer to the administration/deliveryof MYOT miRNAs to a recipient cell either in vivo or in vitro, via areplication-deficient rAAV of the invention resulting in expression of aMYOT miRNA by the recipient cell.

Thus, the invention provides methods of administering an effective dose(or doses, administered essentially simultaneously or doses given atintervals) of rAAV that encode MYOT miRNAs to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows target sites in the myotilin sequence (SEQ ID NO: 11266)for exemplified miRNAs.

FIG. 2 sets out sequences of two MYOT-targeted miRNAs. In each panel,the top sequences indicate the DNA templates from which each respectivemiRNA is transcribed. In the top panel, the DNA template miMYOT.1321 isSEQ ID NO: 2. In the bottom panel, the DNA template miMYOT.1366 is SEQID NO: 3. The folded miRNA transcripts are shown as hairpin structures.The miMYOT.1321 folded miRNA is SEQ ID NO: 11268. The miMYOT.1366 foldedmiRNA is SEQ ID NO: 11271. The mature miMYO.1321 (SEQ ID NO: 11270 whichpairs with SEQ ID NO: 11269 in the figure) and miDUX4.1366 (SEQ ID NO:11273 which pairs with SEQ ID NO: 11272 in the figure) sequences arisefollowing processing in target cells by host miRNA processing machinery(including Drosha, DGCR8, Dicer, and Exportin-5). Sequences shaded ingray indicate sites used for cloning each miRNA into the U6T6 vector.The nucleotides corresponding to the mature miRNA antisense guide strandthat ultimately helps catalyze cleavage of the MYOT target mRNA areunderlined in the miRNA hairpin portions of this diagram. The gray andblack arrowheads indicate Drosha- and Dicer-catalyzed cleavage sites,respectively. The numbers 13, 35, 53, and 75 are provided fororientation. The sequences between (and including) positions 35-53 arederived from the natural human mir-30a sequence, except the A atposition 39, which is a G is the normal mir-30a sequence. This waschanged to an A to facilitate folding of the miRNA loop, based on insilico RNA folding models. The base of the stem (5′ of position 13 and3′ of position 75) is also derived from mir-30a structure and sequencewith some modifications depending on the primary sequence of the guidestrand. Specifically, the nucleotide at position 13 can vary to helpfacilitate a required mismatched between the position 13 and 75nucleotides. This bulged structure is hypothesized to facilitate properDrosha cleavage.

FIG. 3 shows the effect of MYOT-targeted miRNAs in LGMD1A miceexpressing mutant myotilin (MYOT). FIG. 3A is a Western blot showingknockdown of mutant mytotilin expression is muscle extracts fromthree-month old LGMD1A mice, where Left (L)=miMYOT treatment side andRight (R)=miGFP control treated side. FIG. 3B shows real-time PCRresults confirming the Western data.

FIG. 4 shows AAV.miMYO.1321 (labeled miMYOT in the figure) improveshistopathology and muscle weight in 3-mo old TgT57I mice. FIG. 4A. AAVvectors used in 3-mo studies. The miMYOT and control miGFP RNAs areexpressed from the mouse U6 promoter. Both vectors contain a CMV.hrGFPreporter gene cassette. Red rectangles indicated AAV inverted terminalrepeats (ITRs). FIG. 4B. Representative serial sections from T57I miceinjected with AAV.miMYOT (top panels) or AAV.miGFP (bottom panels)controls show reductions in MYOT-seeded protein aggregates. Red spotsare protein aggregates stained by immunofluorescence with MYOTantibodies. Middle panels show overlay with H&E-stained serial sections.Arrows indicate fibers containing centrally-located myonuclei. Rightpanels, aggregates are visible as dark blue spots within the myofiber inserial sections stained with Gomori's Trichrome, while nuclei arepurple. Scale bar, 50 μm. Images shown are representative of 8independently injected animals per virus. FIG. 4C. Quantification ofaggregate staining 3 months after injecting TgT57I GAS muscles withAAV.miMYOT or AAV.miGFP. MYOT knockdown significantly reduced theaverage area of MYOT-positive aggregates by 69% (N=5 muscles per group;5 randomly sampled fields per muscle; paired t-test, p=0.0069; errorsbars represent s.e.m.) FIG. 4D. Graphs show the distribution and averagesize of TgT57I and wild-type (WT) muscles treated with AAV.miMYOT orAAV.miGFP controls, 3 months post-injection. MYOT knockdown in TgT57Imuscles significantly improved myofiber diameter by 4.9 microns (54.8 μmversus 49.9μm in control-treated TgT57I mice; t-test, p=0.047). WT fiberdiameters were 57 and 57.7 microns, in miMYOT- and miGFP-treatedanimals, respectively. N=5 muscles per group; 5 randomly selected fieldsper muscle; an average of 1,205 fibers counted per wild-type animals and1,433 fibers per TgT57I animal). (e) AAV.miMYOT significantly improvedGAS muscle weight by 9.5 mg in 3-mo old TgT57I mice (t-test, p<0.001;N=12 muscles per group). AAV.miMYOT treated muscles averaged 134.4 mg inweight versus 124.9 mg in AAV.miGFP-treated animals; WT controls:miMYOT, 136.0 mg; miGFP, 140.8 mg). (f) The milddegeneration-regeneration effects in TgT57I muscles, as indicated by thepresence of myofibers with centrally-located nuclei, were significantlyimproved 2.1-fold with AAV.miMYOT treatment compared to controls(t-test, p=0.0004). Both group of TgT57I mice were still significantlydifferent from respective WT controls (t-test, p<0.006). *, indicatessignificant difference between miMYOT- and miGFP-treated TgT57I animals.Wild-type animals were not significantly different from one another byall measures, regardless of treatment.

FIG. 5 shows AAV.miMYO.1321 (labeled miMYOT in the figure) improveshistopathology and muscle weight in 9-mo old TgT57I mice. FIG. 5A. AAVvectors used in 9-mo studies. The miMYOT and control miLacZ RNAs areexpressed from the mouse U6 promoter. Both vectors contain a CMV.eGFPreporter gene cassette. Red rectangles indicated AAV inverted terminalrepeats (ITRs). FIG. 5B. Representative serial sections from T57I miceinjected with AAV.miMYOT (top panels) or AAV.miLacZ (bottom panels)controls show reductions in MYOT-seeded protein aggregates. Red spotsare protein aggregates stained by immunofluorescence with MYOTantibodies. Middle panels show overlay with H&E-stained serial sections.Arrows indicate fibers containing centrally-located myonuclei. Rightpanels, aggregates are visible as dark blue spots within the myofiber inserial sections stained with Gomori's Trichrome, while nuclei arepurple. Scale bar, 50 μm. Images shown are representative of 8independently injected animals per virus. FIG. 5C. Quantification ofaggregate staining 9 months after injecting TgT57I GAS muscles withAAV.miMYOT or AAV.miLacZ. MYOT knockdown significantly reduced theaverage area of MYOT-positive aggregates by 52% (N=5 muscles per group;5 randomly sampled fields per muscle; paired t-test, p=0.0085; errorsbars represent s.e.m.) FIG. 5 D. Graphs show the distribution andaverage size of TgT57I and wild-type (WT) muscles treated withAAV.miMYOT or AAV.miLacZ controls, 9 months post-injection. MYOTknockdown in TgT57I muscles significantly improved myofiber diameter by9.1 microns (54 μm versus 44.9 μm in control-treated TgT57I mice;t-test, p=0.0006). WT fiber diameters were 62.5 and 62.2 microns, inmiMYOT- and miLacZ-treated animals, respectively. These values weresignificantly larger than either TgT57I group (p<0.001, t-test). N=5muscles per group; 5 randomly selected fields per muscle; an average of993 fibers counted per wild-type animals and 1,554 fibers per TgT57Ianimal). (e) AAV.miMYOT significantly improved GAS muscle weight by 15mg in 9-mo old TgT57I mice (t-test, p=0.002; N=8 muscles per group).AAV.miMYOT treated muscles averaged 116 mg in weight versus 101 mg inAAV.miLacZ-treated animals; WT controls: miMYOT, 148 mg; miGFP, 154 mg).(f) The mild degeneration-regeneration effects in TgT57I muscles, asindicated by the presence of myofibers with centrally-located nuclei,were significantly improved 2.1-fold with AAV.miMYOT treatment comparedto controls (t-test, p=0.0004). Both group of TgT57I mice were stillsignificantly different from respective WT controls (t-test, p<0.0001).*, indicates significant difference between miMYOT- and miLacZ-treatedTgT57I animals. Wild-type animals were not significantly different fromone another by all measures, regardless of treatment.

FIG. 6 shows AAV.miMYO.1321 (labeled miMYOT in the figure) significantlyimproves whole muscle strength in TgT57I mice 9 months after treatment.AAV.miMYOT-treated TgT57I GAS muscles showed statistically significant38% and 25% improvements in absolute force (FIG. 6A) and specific force(FIG. 6B) compared to AAV.miLacZ-treated controls (N=6-8 legs; p=0.02for (a) and p=0.0009 for (b), t-test). Both TgT57I groups weresignificantly weaker than their WT counterparts (p<0.0001, t-test),while wild-type groups were not significantly different from oneanother.

EXAMPLES

Aspects and embodiments of the invention are illustrated by thefollowing examples. Example 1 describes miRNAs specific for the MYOTgene. Example 2 describes the effect of the miRNAs on the expression ofMYOT as measured by real-time PCR. Example 3 describes rAAV encoding themiRNAs. Example 4 describes the effect of the U6T6 expressing the miRNAson the expression of MYOT as measured by Western blot. Example 5describes delivery of MYOT miRNA to newborn mice. Example 6 describesdelivery of MYOT miRNA to adult mice.

Example 1 MicroRNAs Specific for the MYOT Gene

Six DNAs encoding miRNAs specific for the MYOT gene were generated byPCR. The PCR primers used had the following sequences.

Primer 775 (miMyoT-592-Forward) (SEQ ID NO: 11274):AAAACTCGAGTGAGCGACCTGATTACAATAGCAGTAAACTGTAAAGCCAC AGATGGGPrimer 776 (miMyoT-592-Reverse) (SEQ ID NO: 11275):TTTTACTAGTAGGCAGCCTGATTACAATAGCAGTAAACCCATCTGTGGCT TTACAGPrimer 777 (miMyoT-1291-Forward) (SEQ ID NO: 11276):AAAACTCGAGTGAGCGACTGGATGTCCTTGCAAAAGAACTGTAAAGCCAC AGATGGGPrimer 778 (miMyoT-1291-Reverse) (SEQ ID NO: 11277):TTTTACTAGTAGGCAGCTGGATGTCCTTGCAAAAGAACCCATCTGTGGCT TTACAGPrimer 779 (miMyoT-1321-Forward) (SEQ ID NO: 11278):AAAACTCGAGTGAGCGCGCACCAATGTTTATCTACAAACTGTAAAGCCAC AGATGGGPrimer 780 (miMyoT-1321-Reverse) (SEQ ID NO: 11279):TTTTACTAGTAGGCAAGCACCAATGTTTATCTACAAACCCATCTGTGGCT TTACAGPrimer 781 (miMyoT-1366-Forward) (SEQ ID NO: 11280):AAAACTCGAGTGAGCGAGGAGATTCAGTGAAACTAGAACTGTAAAGCCAC AGATGGGPrimer 782 (miMyoT-1366-Reverse) (SEQ ID NO: 11281):TTTTACTAGTAGGCAGGGAGATTCAGTGAAACTAGAACCCATCTGTGGCT TTACAGPrimer 783 (miMyoT-1490-Forward) (SEQ ID NO: 11282):AAAACTCGAGTGAGCGCGAAGAGTTACTTTACTGATAACTGTAAAGCCAC AGATGGGPrimer 784 (miMyoT-1490-Reverse) (SEQ ID NO: 11283):TTTTACTAGTAGGCAGGAAGAGTTACTTTACTGATAACCCATCTGTGGCT TTACAGPrimer 785 (miMyoT-1603-Forward) (SEQ ID NO: 11284):AAAACTCGAGTGAGCGAGCACGTCCAAACCAAACTCTTCTGTAAAGCCAC AGATGGGPrimer 786 (miMyoT-1603-Reverse) (SEQ ID NO: 11285):TTTTACTAGTAGGCAGGCACGTCCAAACCAAACTCTTCCCATCTGTGGCT TTACAG

DNA encoding a miRNA designated miMyoT-592 was generated using primers775 and 776. DNA encoding miRNA designated miMyoT-1291 was generatedusing primers 777 and 778. DNA encoding miRNA designated miMyoT-1321 wasgenerated using primers 779 and 780. DNA encoding miRNA designatedmiMyoT-1366 was generated using primers 781 and 782. DNA encoding miRNAdesignated miMyoT-1490 was generated using primers 783 and 784. DNAencoding miRNA designated miMyoT-1603 was generated using primers 785and 786. The DNAs are set out below, wherein the number in the namesindicates the 5′ target nucleotide in the myotylin sequence (SEQ ID NO:11267). See FIG. 1 where the target sequences for the miRNAs in themyotilin sequence are underlined.

miMyoT-592 (SEQ ID NO: 5)CTCGAGTGAGCGACCTGATTACAATAGCAGTAAACTGTAAAGCCACAGATGGGTTTACTGCTATTGTAATCAGGCTGCCTACTAGA  miMyoT-1291 (SEQ ID NO: 1)CTCGAGTGAGCGACTGGATGTCCTTGCAAAAGAACTGTAAAGCCACAGATGGGTTATTTTGCAAGGACATCCAGCTGCCTACTAGA  miMyoT-1321 (SEQ ID NO: 2)CTCGAGTGAGCGCGCACCAATGTTTATCTACAAACTGTAAAGCCACAGATGGGTTTGTAGATAAACATTGGTGCTTGCCTACTAGA  miMyoT-1366 (SEQ ID NO: 3)CTCGAGTGAGCGAGGAGATTCAGTGAAACTAGAACTGTAAAGCCACAGATGGGTTCTAGTTTCACTGAATCTCCCTGCCTACTAGA  miMyoT-1490 (SEQ ID NO: 4)CTCGAGTGAGCGCGAAGAGTTACTTTACTGATAACTGTAAAGCCACAGATGGGTTATCAGTAAAGTAACTCTTCCTGCCTACTAGA  miMyoT-1603 (SEQ ID NO: 6)CTCGAGTGAGCGAGCACGTCCAAACCAAACTCTTCTGTAAAGCCACAGATGGGAAGAGTTTGGTTTACGTGCCTGCCTACTAGA 

FIG. 2 shows the DNA templates miMyoT.1321 and miMyoT.1366 and theircorresponding folded and mature miRNAs.

One μg of each primer was added to a 1 cycle primer extension reaction:95° C. for 5 min.; 94° C. for 2 min.; 52° C. for 1 min.; 72° C. for 15min.; and then holding at 4° C. The PCR products were cleaned up withthe Qiagen QIAquick PCR Purification kit before being digested overnightwith XhoI and SpeI restriction enzymes. The digestion product was thenrun on a 1.5% TBE gel and the band excised and purified using the QiagenQIAquick Gel Extraction Kit.

The PCR products were ligated to a U6T6 vector (via XhoI and XbaI)overnight. This vector contains a mouse U6 promoter and an RNApolymerase III termination signal (6 thymidine nucleotides). miRNAs arecloned into XhoI+XbaI restriction sites located between the 3′ end ofthe U6 promoter and the termination signal (SpeI on the 3′ end of theDNA template for each miRNA has complementary cohesive ends with theXbaI site). The ligation product was transformed into chemicallycompetent E-coli cells with a 42° C. heat shock and incubated at 37° C.shaking for 1 hour before being plated on kanamycin selection plates.The colonies were allowed to grow overnight at 37°. The following daythey were mini-prepped and sequenced for accuracy.

Example 2 Real-Time PCR Reaction for Effect of Expression of MYOT miRNAs

Expression of the MYOT target sequence in the presence of the MYOTmiRNAs was assayed. A lipofectamine 2000 transfection was done in C2C12cells in a 12-well, white-walled assay plate. 52,000 cells weretransfected with 100 ng of AAV-CMV-mutMyoT and 1500 ng of one of theU6T6 vectors described in Example 1 containing miRNA-encoding DNA. Theassay was performed 48 hours later.

The media was removed from the cells and 1 μl of Trizol was added perwell. Then the cells were resuspended and the lysates were transferredto 1.5 ml EP tubes. Samples were incubated at room temperature for 5 minand 200 μl chloroform was added. The tubes were shaken vigorously for 15sec, incubated at room temperature for 3 min and centrifuged at 12,000 gfor 15 min at 4° C. Then the aqueous phase was transferred to a freshtube and 0.5 ml isopropyl alcohol was added. The samples were incubatedat room temperature for 10 min and centrifuged at 12,000 g for 10 min at4° C. The RNA pallet was washed once with 1 ml 75% ethanol and aireddry. 20 μl of RNase-Free water was added to dissolve the pellet and theconcentration/purification were measured by Nano-drop. 1.5 ug total RNAwas added to cDNA generation reaction: 5° C. for 10 min.; 37° C. for 120min.; 85° C. for 5 sec and then holding at 4° C. The cDNA products werediluted at 1:10 and 4.5 μl was added to real-time PCR reaction. HumanMyotilin was used to check the expression of the MYOT and the relativeexpression was normalized to mouse GAPDH expression.

U6T6-miMyoT-592 (SEQ ID NO: 5) showed higher expression of MYOT thanU6T6-miGFP control. U6T6-miMyoT-1291 (SEQ ID NO: 1) reduced theexpression of MYOT to 60%, U6T6-miMyoT-1321 (SEQ ID NO: 2) reduced theexpression of MYOT to 19%, U6T6-miMyoT-1366 (SEQ ID NO: 3) reduced theexpression of MYOT to 41.7%, U6T6-miMyoT-1490 (SEQ ID NO: 4) reduced theexpression of MYOT to 55.3%, U6T6-miMyoT-1603 (SEQ ID NO: 6) reduced theexpression of MYOT to 34.9%, when compared to U6T6-miGFP control.

Example 3 Production of rAAV Encoding MYOT MicroRNAs

The U6-miMYOT DNAs were cut from U6T6-miMYOT constructs at EcoRI sitesand then respectively cloned into AAV6-hrGFPs to generate rAAV-U6-miMyoTvectors. These rAAV vectors express miRNA and hrGFP

Example 4 Western Blot Assay for Effect of Expression of MYOT miRNAsfrom U6T6 Vectors and rAAV

The effect of expression of MYOT miRNAs from the U6T6 vectors describedin Example 1 and the rAAV described in Example 3 was assayed by Westernblot.

One day before transfection, 293 cells were plated in a 24-well plate at1.5×10⁵ cells/well. The cells were then transfected with U6T6-miMyoT(592, 1291, 1321, 1366, 1490 or 1603) using Lipofectamine 2000(Invitrogen, Cat. No. 11668-019).

Forty-eight hours after transfection, cells were collected and washedwith cold PBS once. Seventy μl lysis buffer (137 mM NaCl, 10 mM TrispH=7.4, 1% NP40) were then added. The cells were resuspended completelyand incubated on ice for 30 min. The samples were centrifuged for 20 minat 13,000 rpm at 4° C. and the supernatant was collected. The celllysate was diluted 5-fold for the Lowry protein concentration assay(Bio-Rad Dc Protein Assay Reagent A, B, S; Cat. No. 500-0113, 500-0114,500-115). Twenty μg of each sample was taken and 2× sample buffer (100mM Tris pH=6.8, 100 mM DTT, 10% glycerol, 2% SDS, 0.006% bromophenolblue) was added. The samples were boiled for 10 min and then put on ice.

The samples were loaded onto a 10% polyacrylamide gel (based on 37.5:1acrylamide:bis acrylamide ratio, Bio-Rad, Cat. No. 161-0158), 15 μg on agel for each sample. Proteins were transferred to PVDF membranes at 15 Vfor 1 h using semi-dry transfer (Trans-Blot SD Semi-Dry Transfer Cell,Bio-Rad, Cat. No. 170-3940). The blots were placed into blocking buffer(5% non-fat dry milk, 30 mM Tris pH=7.5, 150 mM NaCl, 0.05% Tween-20)and agitated for 1 h at room temperature. The blocking buffer wasdecanted and anti-myotilin primary antibody solution (rabbit polyclonalgenerated by Bethyl Laboratories using a peptide corresponding tomyotilin residues 473-488) was added and incubated with agitationovernight at 4° C. The membranes were then washed for 30 min, changingthe wash buffer (150 mM NaCl, 30 mM Tris pH=7.5, 0.05% Tween-20) every10 min. Peroxidase-conjugated Goat Anti-Mouse Antibody (JacksonImmunoReserch, Cat. No. 115-035-146, 1: 100,000) was added and incubatedat room temperature for 2 h. The membranes were then washed for 30 min,changing the wash buffer every 10 min. The blots were placed inchemiluminescent working solution (Immobilon Weatern ChemiluminescentHRP Substrate, Millipore, Cat. No. WBKLS0500), incubated with agitationfor 5 min at room temperature, and then exposed to X-ray film.

The membranes were washed for 20 min, changing the wash buffer every 10min. Next, stripping buffer (2% SDS, 62.5 mM Tris pH=6.7, 100 mM b-ME)was added to the blots and incubated at 50° C. for 30 min. The membraneswere washed again for 30 min, changing the wash buffer every 10 min.Then, the membranes were blocked again and re-probed with Anti-GAPDHprimary antibody solution (Chemicon, Cat. No. MAB374, 1:200) andperoxidase-conjugated Goat Anti-Mouse Antibody (Jackson ImmunoReserch,Cat. No. 115-035-146, 1:100,000) was used as secondary antibody.

The film was scanned and the density ratio of MYOT to GAPDH wascalculated. Compared to U6T6-miGFP control, the expression of MYOT washigher (1.08) in samples of U6T6-miMyoT-592 (SEQ ID NO: 5) and theexpression of MYOT was reduced to 78.9% by U6T6-miMyoT-1291 (SEQ ID NO:1), 50.2% by U6T6-miMyoT-1321 (SEQ ID NO: 2), 60.2% by U6T6-miMyoT-1366(SEQ ID NO: 3), 76.2% by U6T6-miMyoT-1490 (SEQ ID NO: 4), 87% byU6T6-miMyoT-1603 (SEQ ID NO: 6).

U6T6-miMYOT-1321 most effectively knocked down myotilin expression bothin the real-time PCR and western-blot experiments. The knockdown effectby AAV-miMyoT-1321 was also confirmed by western-blot experiment.

Example 5 Delivery to Newborn Mice

The PCR genotype of newborn pups was determined to identify female WT orT571 MYOT mice (using human MYOT primers and Y chromosome primers).Bilateral intramuscular injections of 5×10¹⁰ AAV6.miMYOT-1321 or controlAAV6.miGFP particles per leg in 1-2 day old mice were sufficient tosaturate the lower limb musculature.

Phenotypic correction was then determined initially by histologicalanalyses. Specifically, 3 months after viral delivery, muscles wereharvested and cryopreserved. Ten micron serial cryosections were cut andstained with antibodies to detect myotilin-positive protein aggregatesin T571 myofibers. AAV6.miMYOT-1321 muscles had significantly reducednumbers of aggregates per section compared to AAV6.miGFP or untreatedcontrols. In addition, when AAV6.miMYOT-132-treated muscles did showoccasional aggregates, they were significantly smaller than those seenin control-treated or untreated T571 animals. AAV6.miMYOT-132 treatmentalso improved muscle size deficits relative to the control treatment.

MYOT knockdown was confirmed by Western blot and real-time PCR as shownin FIG. 3. The AAV delivered miMYOT-1321 significantly reduced mutantMYOT protein (FIG. 3A) and mRNA (FIG. 3B) in the muscles.

These results support therapeutic efficacy. Continuing experimentsinclude determining the functional effects of MYOT knockdown in wholemuscles by measuring EDL specific force.

Example 6 Delivery to Adult Mice

The PCR genotype of weanlings is determined, and 3-month old or 9-monthold mice which have significant pre-existing LGMD1A-associated pathologyare chosen for treatment. 5×10¹⁰ AAV6 vectors are delivered to lowerlimb musculature by isolated leg perfusion. Phenotypic correction(including hindlimb grip strength, gross muscle parameters and EDLspecific force are then measured using various methods over thefollowing months.

Male P1 or P2 mice were injected in the lower limbs with 5×10¹⁰ DNAseresistant particles AAV6.miMYOT.1321 or control AAV6.miGFP particles perleg. Muscles were harvested for analysis at 3 months and 9 months ofage. All mouse protocols were approved by the Institutional Animal Careand Use Committee (IACUC) at The Research Institute of NationwideChildren's Hospital.

Imaging and Histology.

In vivo AAV transduction was determined by GFP epifluorescence using afluorescent dissecting microscope (MZ16FA, Leica, Wetzlar, Germany).Dissected muscles were placed in O.C.T. Compound (Tissue-Tek, Torrance,Calif.) and frozen in liquid nitrogen-cooled 2-methylbutane. The blockswere cut onto slides as 10 μm cryosections, and stained with hematoxylinand eosin (H&E; following standard protocols), or anti-MYOT polyclonalantibodies. For MYOT immunohistochemistry, cryosections were fixed inmethanol and blocked in GFTP⁺ buffer (5% normal goat serum, 0.1% piggelatin, 1% BSA, 0.2% Triton X-100, in phosphate-buffered saline).Slides were incubated overnight at 4° C. with MYOT primary antibody(1:400), and then with AlexaFluor-594 conjugated goat anti-rabbitsecondary antibodies (1:500; 1 hour at RT; Molecular Probes, Carlsbad,Calif.). Images were taken from mouse tissue harvested from 3- and9-month old male mice. Muscle cross-sectional fiber diameters andpercentage of myofibers with centrally-located nuclei were determined aspreviously described from five different animals per group (five fieldsper leg).

Contractile Measurements of Gastrocnemius Muscle.

Mice were anesthetized with intraperitoneal injection of Avertin (250mg/kg) with supplemental injections given to maintain an adequate levelof anesthesia during the whole procedure. The gastrocnemius muscle wasexposed and the distal tendon was isolated and cut. The exposed muscleand tendon were kept moist by periodic applications of isotonic saline.Knot was tied at the proximal end of the tendon and the mouse was placedon a heated platform maintained at 37° C. The tendon was tied securelyto the lever arm of a servomotor (6650LR, Cambridge Technology) via thesuture ends. The muscle was then stimulated with 0.2 ms pulses via theperoneal nerve using platinum electrodes. Stimulation voltage and musclelength were adjusted for maximum isometric twitch force (Pt). The musclewas stimulated at increasing frequencies until a maximum force (Po) wasreached at optimal muscle length (Lo). Optimum fiber length (Lf) wasdetermined by multiplying Lo by the gastrocnemius Lf/Lo ratio of 0.45.Total fiber CSA was calculated by dividing the muscle mass (mg) by theproduct of muscle fiber length (mm) and the density of mammalianskeletal muscle, 1.06 g/cm2. Specific Po (N/cm2) was calculated bydividing Po by total fiber CSA for each muscle. Immediately after musclemass was measured, muscles were coated in tissue freezing medium(Triangle Biomedical Sciences, Durham, N.C.), frozen in isopentanecooled by dry ice, and stored at −80° C. until needed.

EDL Muscle Contractile Measurements (Supplemental Data).

The EDL muscle was completely removed from the animal and the proximaland distal tendons of the muscle were tied with suture. The muscle wasimmersed in a bath containing Krebs' mammalian Ringer solution with 0.25mM tubocurarine chloride. The solution was maintained at 25° C. andbubbled with 95% O₂ and 5% CO₂. The distal tendon was attached to aservomotor (model 305B, Aurora Scientific, Aurora, ON). The proximaltendon was attached to a force transducer (model BG-50, KuliteSemiconductor Products, Leonia, N.J.). The muscle was stimulated bysquare-wave pulses delivered by two platinum electrodes connected to ahigh-power biphasic current stimulator (model 701B, Aurora Scientific,Aurora, ON). The voltage of pulses was increased, and optimal musclelength (L_(o)) was subsequently adjusted to produce maximum twitchforce. Muscles were held at L_(o) and stimulus frequency was increaseduntil the P_(o) was achieved. The sP_(o) was determined by dividingP_(o) by the cross-sectional area (CSA). The L_(f)-to-L_(o) ratios of0.44 for EDL muscles was used to calculate L_(f). The physiological CSAof muscles was determined by dividing the mass of the muscle by theproduct of L_(f) and 1.06 g/cm³, the density of mammalian skeletalmuscle.

Statistical Analysis.

All data are expressed as mean±SEM. Statistical analyses were performedusing the GraphPad Prizm software package. Statistical tests used foreach experiment, and accompanying N's, are indicated in the FigureLegends.

MYOT knockdown improved histopathology and muscle weight in 3-month(3-mo) old TgT57I mice

TgT57I mice recapitulate the progressive MYOT protein aggregationdefects that characterize LGMD1A. In 3 mo-old TgT57I mice, aggregatesare associated with additional generalized muscle pathology, includingdeficits in myofiber size and gastrocnemius muscle weight, as well asslight but significant increase in myofibers with centrally locatednuclei, which is a histological indicator that muscles underwentdegeneration and were subsequently repaired. Importantly, thesephenotypes are useful outcome measures for RNAi therapy. We thereforeexamined the effects of miMYOT-mediated MYOT gene silencing on aggregateformation, myofiber diameter, muscle weight, and central nuclei defectsassociated with LGMD1A in young adult TgT57I mice.

Aggregate accumulation was examined by staining AAV6.miMYOT- andAAV6.control-treated TgT57I gastrocnemius muscle cryosections with MYOTimmunoreactive antibodies, trichrome, and hematoxylin and eosin (H&E)(FIGS. 4A and B). Microscopic image analysis showed that MYOT knockdownsignificantly reduced the abundance of protein aggregates by 69% in 3-moold TgT57I gastrocnemius muscles (FIGS. 4B and C).

Next, the impact of MYOT inhibition on cross-sectional myofiber size wasdetermined using H&E stained muscle cryosections. Myofibers fromAAV.control-treated TgT57I muscles were significantly smaller (49.9 μmaverage diameter; p<0.05) than those from either wild-type group (57.0μm and 57.7 μm in wild-type mice receiving miMYOT or miGFP,respectively; FIG. 4D). In contrast, MYOT knockdown by our therapeuticAAV6.miMYOT vectors improved average myofiber diameter in TgT57I mice by4.9 μm (a 9.8% improvement), to levels not significantly different thanwild-type (54.8 μm in AAV6.miMYOT-treated TgT57I mice; FIG. 4D). Thisimprovement in myofiber size defects evident at the cellular leveltranslated to whole muscle as well. Indeed, weights ofAAV6.miMYOT-treated TgT57I gastrocnemius muscles were not significantlydifferent than those measured in wild-type treated controls, whileTgT57I muscles that received control AAV6.miGFP vector weighed anaverage of 15.9 mg less (11% decrease) than their wild-type counterparts(p<0.001; FIG. 4E). Finally, comparing the AAV6.miMYOT- andAAV6.miGFP-treated TgT57I animals, that MYOT knockdown improved 3-moTgT57I gastrocnemius muscle weight by an average of 9.5 mg, representinga significant 7.1% improvement (p<0.001).

As a final measure of the effects of MYOT knockdown on LGMD1A-associatedhistopathology in 3-mo old TgT57I mice, the percentage of myofiberscontaining centrally-located nuclei was quantified. Typically ˜98-99% ofmyonuclei in uninjured wild-type muscles are localized to the cellperiphery. Consistent with this, gastrocnemius muscles from ourAAV6.miMYOT- and AAV6.miGFP-treated wild-type animals showed 1.1% and1.9% central nuclei, respectively. In contrast, 7.7% of 3-mo TgT57Imyofibers from control AAV6.miGFP-treated gastrocnemius musclescontained central nuclei. This value is consistent with milddegeneration and regeneration in dystrophic animals. Importantly, MYOTknockdown by AAV6.miMYOT reduced the percentage of myofibers withcentral nuclei to 3.6% in TgT57I mice, representing a significant2.1-fold decrease (p<0.001; FIG. 4F).

MYOT knockdown also improves histopathology, muscle weight, and specificforce in 9-mo old TgT57I mice

Gastrocnemius is among the most severely involved muscles in TgT57I miceand LGMD1A patients. Considering this, prospective LGMD1A-targetedtherapies should ideally treat gastrocnemius muscle weakness related tomutant MYOT accumulation. Although 3-mo old TgT57I muscles displayLGMD1A-associated changes in histology and weight, our pilot studiesshowed that significant muscle weakness did not manifest until later inadulthood (9 months of age; data not shown). Therefore, a second cohortof animals were treated with AAV6.miMYO.1321 or control AAV6.miLacZvectors for 9 months, with the goal of correcting whole musclefunctional deficits in aged TgT57I gastrocnemius muscles.

Before measuring specific force, MYOT suppression by AAV6.miMYOT (79%mRNA; 63% protein; FIG. 1 c) was confirmed to be still benefiting TgT57Ianimals at 9-months of age, using the outcome measures established inour younger, 3-mo cohort. AAV6.miMYOT-treated TgT57I animals showedsignificant correction by all measures, compared to AAV6.miLacZcontrol-treated counterparts. Specifically, in 9-mo oldAAV6.miMYOT-treated TgT57I animals, aggregates were reduced by 52%(p<0.01); myofibers were 9.1 μm (20%) larger (54 μm average versus 44.9μm average in AAV6.miLacZ-treated TgT57I; p<O); gastrocnemius musclesweighed 12% more (116 mg average versus 101 mg average inAAV6.miLacZ-treated TgT57I; p>0.002); and central nuclei were reduced1.5-fold (10.6% in AAV6.miMYOT-treated versus 15.5% inAAV6.miLacZ-treated TgT57I; p<0.04). The improvements afforded byAAV6.miMYOT were partial, as TgT57I animals treated with thistherapeutic vector were still significantly different from wild-typegroups using all outcome measures at 9-mos (FIG. 5).

Importantly, MYOT knockdown by AAV6.miMYOT caused significant functionalimprovement in Tg571 gastrocnemius muscles, as determined by wholemuscle physiology tests. Specifically, MYOT knockdown improved absoluteand specific force in 9-mo TgT57I gastrocnemius muscles by 38% and 25%,respectively (FIG. 5). As with the other outcome measures describedabove, this represented a partial functional recovery, as both groups ofTgT57I animals were significantly different from their wild-type treatedcounterparts (FIG. 6).

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the claims should be placed on the invention.

All documents referred to in this application are hereby incorporated byreference in their entirety.

We claim:
 1. A recombinant adeno-associated virus comprising the MYOTmiRNA-encoding DNA set out in SEQ ID NO: 1, 2, 3 or 4, wherein therecombinant adeno-associated virus lacks rep and cap genes.
 2. Acomposition comprising the recombinant adeno-associated virus ofclaim
 1. 3. A method of inhibiting expression of the MYOT gene in a cellcomprising contacting the cell with a recombinant adeno-associated virusof claim 1 or the composition of claim
 2. 4. A method of delivering theMYOT miRNA-encoding DNA set out in SEQ ID NO: 1 to an animal in needthereof, comprising administering to the animal a recombinantadeno-associated virus comprising the MYOT miRNA-encoding DNA set out inSEQ ID NO: 1, wherein the recombinant adeno-associated virus lacks repand cap genes.
 5. A method of delivering the MYOT miRNA-encoding DNA setout in SEQ ID NO: 2 to an animal in need thereof, comprisingadministering to the animal a recombinant adeno-associated viruscomprising the MYOT miRNA-encoding DNA set out in SEQ ID NO: 2, whereinthe recombinant adeno-associated virus lacks rep and cap genes.
 6. Amethod of delivering the MYOT miRNA-encoding DNA set out in SEQ ID NO: 3to an animal in need thereof, comprising administering to the animal arecombinant adeno-associated virus comprising the MYOT miRNA-encodingDNA set out in SEQ ID NO: 3, wherein the recombinant adeno-associatedvirus lacks rep and cap genes.
 7. A method of delivering the MYOTmiRNA-encoding DNA set out in SEQ ID NO: 4 to an animal in need thereof,comprising administering to the animal a recombinant adeno-associatedvirus comprising the MYOT miRNA-encoding DNA set out in SEQ ID NO: 4,wherein the recombinant adeno-associated virus lacks rep and cap genes.8. A method of treating limb girdle muscular dystrophy type 1Acomprising administering a recombinant adeno-associated virus comprisingthe MYOT miRNA-encoding DNA set out in SEQ ID NO: 1, wherein therecombinant adeno-associated virus lacks rep and cap genes.
 9. A methodof treating limb girdle muscular dystrophy type 1A comprisingadministering a recombinant adeno-associated virus comprising the MYOTmiRNA-encoding DNA set out in SEQ ID NO: 2, wherein the recombinantadeno-associated virus lacks rep and cap genes.
 10. A method of treatinglimb girdle muscular dystrophy type 1A comprising administering arecombinant adeno-associated virus comprising the MYOT miRNA-encodingDNA set out in SEQ ID NO: 3, wherein the recombinant adeno-associatedvirus lacks rep and cap genes.
 11. A method of treating limb girdlemuscular dystrophy type 1A comprising administering a recombinantadeno-associated virus comprising the MYOT miRNA-encoding DNA set out inSEQ ID NO: 4, wherein the recombinant adeno-associated virus lacks repand cap genes.
 12. The recombinant adeno-associated virus of claim 1wherein expression of the miRNA-encoding DNA is under the control of aCMV promoter, a muscle creatine kinase (MCK) promoter, an alpha-myosinheavy chain enhancer-/MCK enhancer-promoter (MHCK7) or a desminpromoter.
 13. The composition of claim 2 wherein expression of themiRNA-encoding DNA is under the control of a CMV promoter, a musclecreatine kinase (MCK) promoter, an alpha-myosin heavy chainenhancer-/MCK enhancer-promoter (MHCK7) or a desmin promoter.
 14. Themethod of any one of claims 3-11 wherein expression of themiRNA-encoding DNA is under the control of a CMV promoter, a musclecreatine kinase (MCK) promoter, an alpha-myosin heavy chainenhancer-/MCK enhancer-promoter (MHCK7) or a desmin promoter.